Results And Discussion

Endothelial tip cells selectively express laminin α4

In situ hybridization showed restricted expression of Lama4 at the growing vascular front of the postnatal mouse retina (Fig 1A, red arrowheads), with most abundant expression in the leading tip cells (Fig 1A,B). In comparison, expression of Lama5 is prominent in the developing vascular plexus (Fig 1C,D). Laminin α4 protein is selectively distributed along the abluminal BM of all retinal vessels and appears most tightly associated with the endothelium (Fig 1E). Laminin α5 protein decorates the endothelium, but also retinal astrocytes ahead of and around the newly forming vessels (Fig 1F). Notably, laminin α1 was not detected in the retinal vasculature (supplementary Fig S1 online). The observation of strongest expression of mRNA Lama4 in the leading tip cells indicates that tip cells not only break down BM but also actively produce nascent BM components with a specific function during sprout patterning. In addition, a recent microarray study of Dll4 heterozygous versus wild‐type (WT) retinas identified high expression of several genes involved in the degradation and production of BM in endothelial tip cells (del Toro et al, 2010).

Whole‐mount preparations and sections of the leading vascular front illustrate fragmented laminin α4 staining along the abluminal membrane of tip cells. Only the filopodia of tip cells lack laminin α4 staining (Fig 1G,H). In the plexus, co‐staining with VE‐cadherin and isolectin B4 confirmed the tight association of laminin α4 with the basal and basolateral EC membrane, including areas in immediate proximity to EC junctions (Fig 1I).

As the ability of a given cell to produce Dll4 is controlled in a competitive manner by VEGFR2 levels (Jakobsson et al, 2010), we asked whether Lama4−/− cells could compete with WT cells in mosaic vessels. Chimaeric EBs containing WT DsRed cells and Lama4−/− cells illustrated that Lama4‐deficient cells could form both stalk and tip cells. In stalk position, they expressed less Dll4 (Fig 4H), whereas in tip cell position they expressed abundant Dll4 protein (Fig 4I,J), indicating that neighbouring WT cells can rescue the ability of Lama4−/− cells to produce Dll4. Given the dynamic position shuffling in the sprouts (Jakobsson et al, 2010), it is likely that Lama4−/− cells are exposed to extracellular LN411 produced by WT cells. Curiously, Lama4−/− tip cells were engulfed by WT neighbours such that the Lama4−/− cells contributed minimally to the abluminal surface of the sprout (Fig 4I,J), supporting the idea that WT cells take over the role of BM formation in mosaic vessels.

Loss of laminin α4 phenocopies Notch inhibition

To understand the full physiological relevance of laminin‐α4‐induced Dll4/Notch signalling, we compared EC arrangement and junctional patterning, as well as the formation of vascular lumen, in Lama4‐deficient and DAPT‐treated retinas. Co‐labelling of VE‐cadherin, the EC‐specific transcription factor Erg and isolectin B4 illustrated increased junctional profiles with irregular trajectories, coinciding with clustering of EC nuclei in Lama4−/− compared with control littermate retinas (supplementary Fig S4 online). Immunolabelling of the tight‐junction protein ZO1 confirmed the increased amount and irregularity of EC junctions (supplementary Fig S4 online). Notch inhibition by DAPT treatment led to similar effects (supplementary Fig S5 online). Using intercellular adhesion molecule 2 (ICAM2) as a marker for luminal EC membrane, we observed that most vessels formed lumen in control and Lama4−/− retinas (supplementary Fig S4 online), as well as in DAPT‐treated retinas (supplementary Fig S5 online). However, in mutant retinas and DAPT‐treated retinas, the lumen was more irregular, with wider segments, as well as local narrowing.

Although these results confirm a role of endothelial laminins in vessel stability, diameter control and patterning (Pollard et al, 2006; Jakobsson et al, 2008), we find that Notch inhibition and Lama4 deficiency produce strikingly similar morphological changes, indicating that the modulation of Dll4/Notch signalling is a critical molecular mechanism underpinning the physiological role of laminin α4 in vessel stabilization.

Laminin‐α4‐mediated induction of Dll4 involves integrins

To gain insight into the possible involvement of integrins in laminin‐mediated induction of Dll4, we performed EC (HUVEC) adhesion assays using function‐blocking antibodies to human integrins. Adhesion to LN411 was reduced in the presence of α2 and α3 blocking antibodies (Fig 5A), indicating that both receptors can contribute to EC adhesion to this substrate. Adhesion to LN511 involves a distinct combination of integrins, as α6 and α3 blocking antibodies are more effective in impairing adhesion to this substrate (Fig 5B). To investigate effects on Dll4 induction, we used small interfering RNA (siRNA) to knock down individual integrin subunits (supplementary Fig S6 online). LN411‐induced Dll4 expression was significantly reduced on treatment with siRNA for integrin α2 and α6, but not α3 compared with control siRNA (Fig 5C,D).

To assess the physiological relevance in vivo, we studied retinal angiogenesis in mice with either global or endothelial‐specific deficiency in α2‐, α3‐, β3‐ and β1‐integrin (Fig 5E–Q). None of the mutants deficient for individual α‐subunits or the β3 subunit showed a hypersprouting phenotype reminiscent of the Lama4 mutant (Fig 5L–Q). Endothelial deletion of α6 integrin, similar to β3 integrin deletion, leads to enhanced tumour angiogenesis, but does not seem to affect developmental angiogenesis (Germain et al, 2010). Integrin β1 combines with several α‐subunits expressed in the retina (supplementary Fig S6 online), such as integrin α2β1, α3β1 and α6β1 to form functional heterodimers. Using an endothelial‐specific inducible approach (Pdgfb‐iCre), we asked whether a short‐term induced loss of endothelial‐derived integrin β1 results in reduced Dll4 expression and hypersprouting. Indeed, at 2–3 days post induction of Cre‐recombination by tamoxifen injection, we observed a strong reduction of itgb1 expression (supplementary Fig S6 online) coinciding with reduced Dll4 staining at the vascular front (Fig 5I–K), and a striking peripheral hypersprouting phenotype in Itgb1flox/flox Pdgfb‐iCre (Itgb1 EC−/−) mice (Fig 5E–H). Taken together, these results indicate that several α‐subunits in combination with the β1‐subunit can mediate signalling events triggered by laminin α4 to induced Dll4 and hence regulate tip/stalk patterning.

Immunofluorescence. Eyes were collected at P3 to P7 and fixed in 4% paraformaldehyde for 2 h at room temperature. For whole‐mount preparation, retinas were dissected and vessels were visualized using isolectin B4. For sections, eyes were passed through sucrose/PBS and embedded in TissueTec. For antibody details see supplementary information online. Retinas were analysed by confocal laser scanning microscopy.

In situ hybridization. Mouse Vegfa, Pdgfb and Lama4 complementary DNA probes were digoxigenin labelled and used for whole‐mount retina in situ hybridization as previously described (Fruttiger, 2002).

BrdU labelling. BrdU was injected 2 h before eyes were isolated, fixed and dehydrated. After dissection, retinas were treated with proteinase K and DNaseI as described (Abramsson et al, 2007). ECs were visualized by isolectin B4 and BrdU was detected using mouse anti‐BrdU Alexa 488 (Molecular Probes).

Quantifications. Branchpoints were counted per 400 μm2 visual field. Relative vascular density was determined by measuring isolectin‐B4‐positive surface area in relation to the total vascularized area using ImageJ. Filopodia were counted and normalized to leading vascular membrane length. Statistical significance was verified using t‐test.

Dll4 labelling quantification was performed using Imaris ‘surface’ function on 16‐bit × 40 images of sprouting region of Lama4+/− and Lama4−/− retinas.

Quantitative PCR. Eyes were collected in RNAlater (Qiagen). RNA was isolated from tissues and cells, and quantity and quality of RNA was determined. RNA was converted using SuperScriptIII (Invitrogen), followed by quantitative real‐time PCR. Gene expression assays specific for either mice or humans were from Applied Biosystems.

Cell culture. HUVECs and mouse brain ECs (bEND5) were seeded on plates and coated with 10 μg/ml CollagenIV, LN411, LN511 or with PBS at 4°C. Matrix substrates were aspirated and cells were cultured for up to 72 h.

SDS gel electrophoresis and western blotting. Cell lysates were obtained in RIPA (RadioImmunoPrecipitation Assay) buffer. Equal amounts of protein samples were run on 4–12% Bis‐Tris gradient gel and blotted according to NuPage System manufacturer's instruction. For antibodies used, see supplementary information online.

ES cell generation and embryoid body assay. Two Lama4−/− cell lines were established and used for generation of 3D EBs as described (Jakobsson et al, 2007). DsRed‐MST ES cell line used as control was a kind gift from Dr Andras Nagy.

Adhesion assays with integrin‐blocking antibodies. HUVECs were used in adhesion assays in the presence of integrin‐blocking antibodies against α2 (AK7), α3 (ASC‐1), α6 (GoH3) or anti‐major histocompatibility complex antibody (W6.32) as control.

Knockdown of integrin subunits by siRNA. siRNA transfection was carried out using Magnetofection technology (polyMag; OZ Biosciences). ON‐TARGETplus SMARTpool L‐004571 and L‐007214 (Dharmacon) directed against α3 and α6, respectively, as well as siRNA targeting α2 integrin and luciferase as a control (si Ctrl), were used at a final concentration of 100 nM.

Protein extraction. Protein was extracted 24 h after plating on different extracellular matrices using ice‐cold RIPA buffer. Lysates were analysed by immunoblotting.

FACS analysis of surface‐protein expression. Cells were detached and incubated with antibodies against α2 (AK‐7), α3 (ASC‐1) or α6 (GoH3) integrins, followed by secondary fluorescein isothiocyanate‐conjugated antibody. Cells were fixed in 1% formaldehyde and mean fluorescence intensities were determined.

Conflict of Interest

Supplementary Information

Acknowledgements

We are grateful to Sue Watling, Claire Darnborough and Craig Thrussel for animal care and tissue collection, and Ken Blight and Anne Weston for assistance with en face TEM. We thank Kairbaan Hodivala‐Dilke and Stephen Robinson for α3‐ and β3‐integrin‐deficient mouse retinas, and Beate Eckes and Jan Schulz for α2‐integrin‐deficient mouse retinas. This work was supported by Cancer Research UK, the Lister Institute of Preventive Medicine, a Leducq Transatlantic Network Grant (Artemis), the Max Planck Society, the Collaborative Research Centre SFB 492 of the DFG, and the EMBO Young Investigator Programme. C.A.F. is supported by a Marie Curie Actions Fellowship of the FP7 People Programme. S.E. and A.M. are supported by INSERM, CNRS, Agence Nationale de la Recherche (ANR A05135AS), INSERM‐CDD fellowship to S.E.